Metastasis of tumor cells from the primary site of a malignancy accounts for the majority of fatalities in cancer patients. The process of metastasis is complex and involves several steps including invasion and degradation of the extracellular matrix, intravasation, transit through the vasculature, extravasation, and establishment of new tumor foci distant from the primary tumor. These steps probably involve the actions of several proteolytic enzymes which are regulated at several levels temporally during the metastic process. Through its ability to activate plasminogen to plasmin, uPA is among the enzymes involved in the metastic process. We are using a human osteosarcoma model comprised of cell lines showing varying abilities to form tumors and metastasize in athymic nude mice. The gene encoding uPA is single copy in each of the cell lines. By Northern blot hybridization, the nontumorigenic parental cell line HOS expresses low levels of uPA mRNA while two of its transformed deriatives, AD110 and KRIB, express approximately 5-10 times as much uPA mRNA. Preliminary promoter analysis using CAT assay suggest that the 5' flanking region of uPA contains a negative regulatory element active in HOS cells. The same region appears to confer positive promoter activity in AD110 and KRIB cells. Procedures for isolating nuclei for nuclear run on assays have been optimized and nuclear run on assays are currently underway.